This invention relates to the specific detection of DNA of a specific base sequence. In particular, it relates to the construction of a DNA molecule, termed a "probe-vector", which is complementary to the DNA sequence that one wishes to detect, called the "target" sequence, and which will transform bacteria at high efficiency if and only if it has hybridized with the target sequence.
Deoxyribonucleic acid, or DNA, is a long linear polymer of units called nucleotides. Each nucleotide contains any one of the four nitrogeneous bases adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence of bases in an organism's DNA specifies the genetic characteristics of the organism. Most of the individual organisms belonging to a species share most of their respective DNA sequences in common. Accordingly it is possible to identify DNA sequences which all or most of the individual organisms of a species contain but which do not exist in organisms outside the species. Such a DNA is characteristic of the species, and is in a sense "diagnostic" of it.
The ability to detect and identify particular species has application in the diagnosis of infectious diseases. Various pathogens, for example, viruses, bacteria, fungi, and protozoa, can be detected and identified by detecting particular DNA sequence in clinical specimens by this invention. Further genetic characteristics of an infecting organism which affect the pathogenicity or resistance to therapeutic agents (for example antibiotic resistance) can also be detected and identified by this invention.
Within a species, individual organisms exhibit genetic differences from one another. In some cases these differences are manifested as inherited diseases, such as sickle cell anemia in man. These differences can be detected as differences in the base sequence of the DNA of the various organisms. Other diseases such as diabetes and heart disease have genetically determined predispositions which can be identified by characteristic variations in the DNA sequence of the individual. This invention can be applied to detect and identify these variations, and thereby, the genetic predispositions they indicate.
Rearrangements of genomic DNA can result in sequences which were formerly far away from each other being brought into close proximity. Such genetic transpositions occur during development of the immune system, and are implicated in the etiology of some cancers. The probe-vector of this invention requires close linkage between two target sequences for detection of those sequences. Thus suitable probe-vectors can be used to detect rearranged sequences resulting from genetic transpositions.
Since the characteristic DNA sequence one wishes to identify may (is likely to) be found in the presence of a vast abundance of DNA of different sequence it is necessary that its method of detection be highly specific. Further, since little DNA of the characteristic sequence may be available for analysis, a method of high sensitivity is also desirable.
DNA possesses a fundamental property called base complementarity. In nature DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be apposed to the base thymine (T) on the other strand, and the base guanine (G) will be apposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or "denaturation", of the two strands. If the DNA is then placed in conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or "hybridize", and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only strands with a high degree of base complementarity will be able to form stable double stranded structures. The relationship of the specificity of hybridization to reaction conditions is well known. Thus hybridization may be used to test whether two pieces of DNA are complementary in their base sequences.
Many genera of bacteria harbor DNA molecules called plasmids. Plasmids are circular molecules which are separate from the main set of bacterial genes. Plasmids can be taken up by bacteria under appropriate conditions, in a process called transformation. They contain the sequences necessary to insure their own replication, and commonly, they also contain other sequences giving the bacteria an easily detectable phenotype, such as antibiotic resistance.
Plasmids have been modified in vitro by a variety of biochemical techniques. Most notable among these are the recombinant DNA procedures whereby sections of foreign DNA are inserted into plasmids. This is accomplished with the aid of various enzymes, in particular restriction endonucleases, which cleave DNA at sites determined by specific base sequences, and ligases, which can be used to re-join the ends of DNA. See U.S. Pat. No. 4,237,224 to Cohen et al.
Of fundamental importance to our invention is the fact that in order to efficiently transform a bacterial cell such as Escherichia coli, a plasmid DNA must have a circular configuration. Transformation of E. coli with intact double stranded plasmids containing 2-15 kilobase pairs can proceed with an efficiency on the order of 1.times.10.sup.8 transformed cells per microgram of input DNA (see D. Hanahan, J. Mol. Biol. 166: 557-580, 1983), or one transformed cell per 10.sup.3 DNA molecules for plasmids of about 4 kilobase pairs. In contrast, linear plasmid DNA (that is, formerly circular DNA molecules in which both strands have been cut once at the same point) transforms E. coli very poorly, perhaps one thousand times less well than the same DNA in a circular form. Plasmids may remain circular even if both strands have been cut, if the cut sites are separated by enough base pairs that the interactions between the strands are strong enough to hold the two cut strands together. Such cut, but still circular, plasmids transform almost as efficiently as uncut molecules (see D. Hanahan, supra).
Circular single stranded DNA molecules exist in nature as the genomes of certain viruses. These DNAs can also enter and establish within E. coli cells, but with decreased efficiency (around 1/10th as well as otherwise equivalent double stranded circles). Linear single stranded forms of plasmids transform E. coli with efficiencies so low as to be difficult to quantify.
The invention described herein combines the specificity of DNA hydridization with the sensitivity of bacterial transformation to yield a method for the specific and sensitive detection of DNA sequences. An additional benefit of this method is that it is possible, with the appropriate DNA reagents, to clone a portion of the sequence being detected. This permits further study of the DNA by methods such as sequencing and restriction enzyme cleavage.